Use of in-situ atomic force microscopy to monitor the biodegradation of polyhydroxyalkanoates (PHAs)

Abstract

Thin films of a co-polymer mixture of poly 3-hydroxybutyrate and poly 3-hydroxyvalerate P(3HB-3HV) were spun-cast onto glass slides resulting in 35 nm thick layers with a spherulitic microstructure. An untyped strain of Streptomyces sp. bacteria was isolated from soil samples, and it's PHA depolymerase was used to degrade the P(3HB-3HV) thin films. Both ex-situ and in-situ atomic force microscopy (AFM) biodegradation studies were performed to determine the kinetics of the biodegradation over the course of three hours at room temperature. Ex-situ AFM was performed in Tapping Mode and in-situ AFM was performed in the PHA depolymerase using contact mode AFM in the liquid cell, allowing for the real-time analysis of P(3HB-3HV) biodegradation. Biodegradation is observed uniformly throughout the surface, and can be observed within 30 min. of depolymerase exposure. In-situ AFM analysis yields a linear degradation rate as a function of time, while the ex-situ study suggests a more complex kinetics.